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Review Carlos Quesada-Gómez1,2 Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates 1 2 Laboratorio de Investigación en Bacteriología Anaerobia, Centro de Investigación en Enfermedades Tropicales, Universidad de Costa Rica Sección de Bacteriología General, Facultad de Microbiología, Universidad de Costa Rica. ABSTRACT The conjugation is one of the most important mechanisms of horizontal gene transfer in prokaryotes, leading to genetic variation within a species and the acquisition of new traits, such as antibiotic resistance. Bacteroides is an obligate anaerobe of the colon and a significant opportunistic pathogen. Antibiotic resistance among Bacteroides spp. is rapidly increasing, largely due to the dissemination of DNA transfer factors (plasmids and transposons) harbored by members of this genus. Transfer factors can be divided into two classes, conjugative and mobilizable. Species of the intestinal Bacteroides have yielded different resistance plasmids, all of which have been intensely studied, the plasmids encode high-level MLS resistance conferred by a conserved erm gene. It has been reported an interesting observation associated with the transfer of several of these types of elements, all of which conferred Tcr and displayed greatly increased transfer efficiency following exposure to tetracycline. Many of the conjugative transposons (CTns) in Bacteroides are related to various genetic elements (such as CTnDOT, CTnERL, NBU and others). CTnDOT carries a tetracycline resistance gene, tetQ, and an erythromycin resistance gene, ermF. Resistance to drugs used to treat Bacteroides infections, such as clindamycin, has also been increasing. These conjugal elements have been found in Bacteroides clinical isolates. Thus, horizontal gene transfer could conceivably have played a role in the rising incidence of resistance in this bacterial group. Keywords: antimicrobial resistance, Bacteroides , conjugative transposons Correspondence: Carlos Quesada-Gómez Laboratorio de Investigación en Bacteriología Anaerobia, Facultad de Microbiología, Universidad de Costa Rica. 2060, San Pedro, Montes de Oca, San José, Costa Rica. E-mail: [email protected] 184 Elementos genéticos conjugativos y mobilizables en Bacteroides: resistencia a los antibióticos en aislamientos clínicos RESUMEN La conjugación es uno de los mecanismos más importantes de la transferencia horizontal de genes en procariotas, lo que lleva a la variación genética dentro de una especie y la adquisición de nuevos rasgos, como la resistencia a los antibióticos. Bacteroides es un anaerobio obligado y un patógeno oportunista importante. La resistencia a los antibióticos entre especies de Bacteroides está aumentando rápidamente, debido en gran parte a la difusión de los factores de transferencia de ADN (plásmidos y transposones) albergado por los miembros de este género. Los factores de transferencia se pueden dividir en dos clases, conjugativos y movilizables. Las especies de Bacteroides han presentado plásmidos de resistencia a los antibióticos, todos los cuales han sido intensamente estudiados. Estos plásmidos codifican de alto nivel de resistencia MLS conferida por un gen erm conservado. Se ha informado una observación interesante asociada a la transferencia de varios de estos tipos de elementos, todo lo cual confiere mayor resistencia y que aparecen en gran medida la eficiencia de transferencia después de la exposición a la tetraciclina. Muchos de los transposones conjugativos (CTn) en Bacteroides están relacionados con varios elementos genéticos (como CTnDOT, CnTnERL, NBU y otros). CTnDOT lleva un gen de resistencia a la tetraciclina, tetQ, y un gen de resistencia a la eritromicina, ermF. La resistencia a los medicamentos utilizados para tratar infecciones por Bacteroides, tales como la clindamicina, también ha ido en aumento. Estos elementos conjugativos han sido encontrados en los aislados clínicos de Bacteroides. Por lo tanto, la transferencia horizontal de genes posiblemente podría jugar un papel importante en la creciente incidencia de la resistencia bacteriana en este grupo. Palabras clave: resistencia antimicrobiana, Bacteroides, transposones conjungativos Rev Esp Quimioter 2011;24 (4): 184-190 24 C. Quesada-Gómez Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates INTRODUCTION plasmids encode high-level MLS resistance conferred by a conserved erm gene6,9,10. Conjugal genetic exchange in Bacteroides was first described in 1979 when reports from three laboratories demonstrated conjugally transmissible macrolidelincosamide-streptogramin (MLS) resistance in this genus. This resistance phenotype was important because it permitted bacterial growth in the presence of clinically effective and widely used antibiotic clindamycin. This resistance phenotype was similar to that seen in Grampositive bacteria (e.g., streptococci, staphylococci and bacilli). The nucleotide sequence of two of the erm genes (ermF from pBF4 and ermFS from pBI136) has been determined, and the genes have been found to differ at only one nucleotide position. Physical analyses of both wild-type and mutant plasmids revealed the erm gene of each plasmid was bordered by a similar, if no identical, sequence, which flanked the gene in a directly repeated fashion. Other than their erm genes and the associated directly repeated DNA sequences, pBF4, pBFTM10 and pBI136 shared no detectable homology11. In these bacteria, the MLS gene (designated erm) encoded and RNA methylase that modified specific adenine residues in the 23S rRNA, thus preventing MLS drugs from binding to the ribosome and inhibiting protein synthesis. The genetic basis of such conjugal transfer has been traced to both plasmids as well a nonplasmid elements. The latter appear to reside chromosomally and to resemble the conjugative transposons of the Gram-positive bacteria in their behavior1. The repeated sequences bordering the erm genes of these three plasmids have been shown to be a related, if no identical, insertion sequence (IS) elements (IS4400 on pBFTM10, IS4351 on pBF4, and iso-IS4351 on pBI136). These flanking IS elements form compound transposons with their associated erm genes, and genetic evidence for their transposition in both B. fragilis and Escherichia coli. In all three of these transposons the erm genes were juxtaposed to one copy of the IS element, suggesting some interaction between these two sequences. This suggestion has been experimentally explored by the analysis of the nucleotide sequence data, S1 nuclease transcription mapping studies, and the construction of gene fusions to evaluate promoter activity. Collectively, these data indicate that the erm genes arer under the transcriptional control of the promotors contained in the IS element1,12,13. The conjugation is one of the most important mechanisms of horizontal gene transfer in prokaryotes, leading to genetic variation within a species and the acquisition of new traits, such as antibiotic resistance or the ability to produce toxins. Bacteroides conjugative transposons (CTn) were among the first transposons discovered, and they have since been shown to play a central role in the dissemination of antibiotic resistance genes in the related genera2. In particular, a family of CTns, exemplified by CTnDOT and CTnERL, appears to be playing an important role in transferring resistance genes among Bacteroides strains. CTns of this family not only transfer themselves but also mobilize coresident plasmids. In addition, proteins encoded on these CTns trigger in trans the excision and circularization of mobilizable integrated elements called NBUs, and they mobilize these circular forms Bacteroides or Escherichia coli recipients3. They may also mobilize other integrated elements that have been given transposon designation, such as Tn4399, Tn4555, Tn55204,5. To distinguish mobilizable elements that have been given a transposon designation from nonmobilizable Bacteroides transposons such as Tn4351 or Tn45516,7,8. BACTEROIDES TRANSPOSONS IN ANTIBIOTIC RESISTANCE PLASMIDS Species of the intestinal Bacteroides have yielded three different resistance plasmids, all of which have been intensely studied: pBF4, a 41-kb plasmid from B. fragilis; pBFTM10, a 15-kb plasmid from B. fragilis (and its indistinguishable counterpart pCP1 from B. thetaiotaomicron); and pBI136, an 80 kb-plasmid from B. ovatus all share some common features (table 1). All three 25 Examples of the IS element being in opposite orientation with respect to the erm gene were found in the comparative examination of pBF4 and pBFTM10 with pBI136. Odelson et al.10 have proposed a model to explain the formation of these transposons tha predicts that all three transposons evolved independently of one another. Following the entry of a similar erm gene into Bacteroides, transposition of the IS element next to the newly acquired gene resulted in the transcriptional control of erm by the IS element. This may have been required in the face of selective pressure because the invading erm gene could not be expressed in Bacteroides for several reasons (e.g., unrecognizable promoter, nofunctional upstream regulatory sequences on the erm mRNA)10. A NEW NONPLASMID CONJUGATIVE ELEMENTS IN BACTEROIDES It has been know since early in the 1980s that certain antibiotic-resistant strains of Bacteroides fragilis were capable of transferring their resistance without the involvement of detectable plasmid DNA. Such transfer elements have been placed in two categories with respect to antibiotic resistance phenotypes: those that transfer tetracycline resistance alone and those which transfer tetracycline and clindamycin resistances in a linked fashion9. Salyers and Shoemaker 12 reported an interesting observation associated with the transfer of several of these Rev Esp Quimioter 2011;24 (4): 184-190 185 C. Quesada-Gómez Table 1 Genetic element Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates Summary of mobilizable and conjugative genetics elements that confers antimicrobial resistance in Bacteroides. Description pBF4, pBFTM10, pBI136 41-kb, 15-kb, 80-kb plasmids. They encode high-level MLS resistance conferred by erm genes Tn5030 ERL-type element. Mobilizable transposon. That encodes tetracycline and MLS resistance Tn4399 Transposon capable of mobilizing plasmids to E. coli recipient. CTnDOT Major conjugative transposon from Bacteroides. That carries tetQ and ermF genes Tn916 Enterococcal CTnDOT-type elements found in Bacteroides isolates NBU Non-replicating Bacteroides units. NBUs, like the CTn that excise and mobilize them, excise to form covalently closed circular intermediates Tn5520, Tn4555 Mobilizable transposons, they rely on the CnTn for transfer. This confers resistance to other antibiotics (such as β-lactamics) CTnBST Conjugative transposon. 100 kbp. This contains ermB gene CTn341 CTn of the CTnDOT family. Major functional genes: DNA metabolism, conjugation and antibiotic resistance. cLV25 Movilizable transposon with similarity to the Tn4399 types of elements, all of which conferred Tcr and displayed greatly increased transfer efficiency following exposure to tetracycline. Specifically, when these elements were transferred to B. uniformis and the resulting strain was exposed to inhibitory tetracycline, two circular DNA forms (10 and 11-kb) could be detected as extrachromosomal elements. Southern analyses indicated these circular molecules were normally integrated into the host cell chromosome. Certain of the Tc r nonplasmid elements examined by these authors did not mediate the excision of circular DNA forms. Stevens et al.13 have presented evidence suggesting that the control of production of these circular DNA forms resides in a region contained within the Tcr transfer element called ERL (confers Tcr and MLS resistance). Halula and Macrina14 have also reported the partial cloning of Tcr, MLSr element, called Tn5030, from B. fragilis. They estimated the size of this element to be in the 40 to 50-kb range, based on overlapping clones. Interpretation of their results was complicated by the unexpected finding that sequences related to Tn5030 were present in the transconjugant strain from wich their genomic libraries were prepared. Further studies are needed to fully define the molecular structure of this element. Tn4399 was capable of mobilizing plasmids in intergeneric Bacteroides matings as well as in Bacteroides-E. coli matings (table 1). This transfer element provides yet another novel example of the genetic plasticity in anaerobic bacteria, and its is likely to play a role in the dissemination of antibiotic resistance determinants2. Other feature is the ability of most Bacteroides CTns to induce conjugation 1000- to 10000-fold in the presence of tetracycline. The transfer of both the CTn and unliked elements is enhaced by growth in the presence tetracycline at concentration of < 1 mg/L, and this induction is mediated by a complex signal transduction pathway that includes a two-component regulatory system (RteA and RteB) and at 186 least one other regulatory protein, RteC 15. The ability to mobilize other genetic elements and the antibioticstimulated transfer may in part explain the high tetracycline resistance and the high frequency of the other antibiotic resistance genes in Bacteroides16. CTnDOT: A BACTEROIDES CONJUGATIVE TRANSPOSON Many of the conjugative transposons (CTns) in Bacteroides are related to an element calle CTnDOT (table 1). CTnDOT carries a tetracycline resistance gene, tetQ, and an erythromycin resistance gene, ermF16. In fact, without tetracycline stimulation of donor cells, virtually no transfer occurs. Any studies identified a central regulatory region on CTnDOT that is required for tetracycline induction of transfer functions. This region contains a threegene operon that consists of the tetracycline resistance gene tetQ and two regulatory genes, rteA and rteB. RteA and RteB are most closely related to the sensor and response regulator components, respectively, of twocomponent regulatory systems. RteA presumably activates RteB, and activated RteB stimulates the expression of excision and transfer genes. The regulatory cascade mediated by RteA, RteB and RteC is not, however, the first step in tetracycline stimulation of CTn transfer in Bacteroides. Expression of the tetQ-rteA-rteB operon itself is controlled by tetracycline. Stevens et al.13 reported that fusion of tetQ to the reporter gene uidA produced fusion whose expression was stimulated by tetracycline. Intercellular transposition of CTns in Bacteroides is thought to occur by the following steps. The CTn initiates conjugal transfer by excising from the chromosome to form a circular intermediate. This intermediate is nicked at the transfer origin (oriT), and a single-strand copy is transferred to the Rev Esp Quimioter 2011;24 (4): 184-190 26 C. Quesada-Gómez Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates recipient cell, recircularized, and integrated into the recipient´s genome15,16. Since CTnDOT appeared to integrate site selectively, the comparions of the sequences of the ends of CTnDOT with the sequences of the integration sites revealed some sequence similarity. One candidate for recognition sequence is a 10-bp sequence that is immediately adjacent to the site where CTnDOT entered the cromosome and at the right end of the circular form of the element. In the integrated form, this target 10-bp sequenced is 5-bp from the left end of CTnDOT from insertions in four other chromosomal sites. Similar 10-bp sequences were seen in all four cases7,15,16. The integration and excision of CTns have been studied in detail only in the case of Tn916, a CTn first found in enterococci. The CTnDOT-type elements differ in a number of ways from Tn916. First, Tn916 integrates relatively randomly whereas CTnDOT appears to integrate site selectively into about seven sites on the Bacteroides chromosome. Second, there is no sequence similarity between CTnDOT and Tn916 in any of the regions so far sequenced. A third difference between CTnDOT and Tn916 is that transfer of CTnDOT is stimulated 1000- to 10000-fold by tetracycline, a fold increase much higher than the tetracycline stimulation reported for Tn916 transfer8. The tetracycline-dependet stimulation of CTnDOT transfer is mediated by proteins encoded by three regulatory genes: rteA, rteB and rteC. No regulatory genes analogous to the rte genes of CTnDOT have been found on Tn91616. REGULATION OF BACTEROIDES OPERON THAT CONTROLS EXCISION AND TRANSFER OF THE CTNDOT Exist a translational attenuation-type regulation of the tetQ-rteA-rteB operon in response to tetracycline. RT-PCR analysis showed that similar amount of the mRNA from this operon is made regardless of whether tetracycline is present result rules out another possible model for attenuational control of gene expression, the binding of tetracycline to a structure in the mRNA itself in such a way as to cause antitermination. In the case of an antitermination type of control, the concentration of tetQ mRNA should have been much higher under inducing conditions. Although tetQ mRNA was made both in the presence and absence of tetracycline stimulation, translation of the message, is much higher in tetracyclinestimulated cells than in cells no stimulated. This observation taken together with the fact that a translational fusion with uidA was responsible for much higher activity and a higher increase in tetracycline-stimulated cella than a transcriptional fusion in the same locus8, supports the idea that regulation occurs at the level of translation rather than transcriptional. Further evidence for translational attenuation came from mutagenesis experiments that were guided but the observation that hairpain structure could form in the leader region of the mRNA. 27 Production of RteA and RteB, the other protein encoded in the tetQ-rteA-rteB operon, should be regulated similarly to production of TetQ. That is, the proteins are produce at detectable levels only when the cells are stimulated with tetracycline. Since rteA and rteB have been shown to be in the same operon as tetQ, production of these proteins is presumably due to translational coupling one ribosomes begin to translate the operon mRNA. RteA and RteB have no role in tetracycline regulation of operon expression. This was somewhat surprising because RteA and RteB are both essential for CTn excision and transfer functions and resemble components of a two-component regulatory system. RteA, the putative sensor protein, is a sensing something other than tetracycline15,16. TetQ was not required for tetracycline stimulation, but its presence did have an effect. Moreover, increased levels of TetQ due increased levels copy numbers of tetQ caused a greater decrease in protein production. TetQ is a ribosome protection type of tetracycline resistance protein, which is most closely related to an elongation factor. TetQ modifies ribosomes so that tetracycline no longer binds as efficiently. The fact that a higher intracellular concentration of TetQ increased the MIC and further decreased the translation of operon mRNA suggets that one copy of tetQ is no sufficient to protect all of the ribosomes in the cell 8. The modulating effect of TetQ on translation may be a means for the cell to limit the production of RteA and RteB and thus the amount of CTn excision and transfer that occurs. Excision of CTnDOT in Bacteroides is apparently not a lethal event because the chromosomal site from which the CTn is excised is resealed in the process16, but too much activity of this sort could have some deleterious effect on fitness that could be important in an enviroment as competitive as the human colon. THE NBUs ELEMENTS Bacteroides conjugative transposons can also mediate the excision and mobilization of unliked integrated elements called NBUs (non-replicating Bacteroides units). The NBUs so far characterized are 10 to 12 kbp in size. NBUs, like the conjugative transposons that excise and mobilize them, excise to form covalently closed circular intermediates that are transferred by conjugation to a recipient, where their integrate into recipient genome17 (table 1). The circular forms of the NBUs appeared not replicate and were designated nonreplicating units to distinguish them for the plasmids. The circular forms of the NBUs were see only when the strain was grown in medium containing tetracycline. Subsequently, we found that the NBUs are normally integrated in the chromosome and that trans action by a Tcr element is requerired to excise and circularize them18. The NBUs are sometimes cotransferred with Tcr elements, and it was postulated that the excised circular form of the NBUs were plasmidlike forms and were transferred like plasmids and the integrated into the recipient chrosomome3,17. The mobilizable element NBU2 (11 kbp) was found Rev Esp Quimioter 2011;24 (4): 184-190 187 C. Quesada-Gómez Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates originally in two Bacteroides clinical isolated, B. fragilis ERL and B. thethaiotamicron DOT. NBU2 appeared to be very similar to NBU1 in a 2,5 kbp internal region, but this identify between NBU1 and NBU2 is limited to this small region. The sequence of NBU2 revealed two ORFs whose derived amino acid sequences were related to those of known antibiotic resistance genes previously found in Gram-positive bacteria. The deduced amino acid sequence of linAN2, LinA(N2), had 50 to 52% identify and 70 to 72% similitary to the LinA´of Staphylococcus aureus and LinA on pIP855 in S. haemolyticus. LinA is an Onucleotidyltransferase which inactives lincosamides including lincomycin and clindamycin. This is also the first sighting of a linA type gene in Bacteroides species3,18. TN4555 AND THE ANTIBIOTIC RESISTANCE IN BACTEROIDES In Bacteroides species are integrated elements that are much smaller than the CTns. These elements, exemplified by NBU1, Tn5520 and Tn4555, have been called mobilizable transposons because they rely on the CTns for transfer functions8 (table 1). The integrases of NBU1, Tn5520 and Tn4555 have been sequenced, and all are members of the lambda family integrases. NBU1 excision more closely resembles excision of phage lambda in that there is an att site formed by the joined ends that integrates into an identical site in the choromosome5. Although the mobilizable transposons rely absolutely on the CTns for transfer, there is so far no evidence for any significant sequence similarity between them and the CTns. Bacteroides are potencial reservoirs of antibiotic resistance genes and Tn4555 from Bacteroides vulgatus carries cfxA, encoding an extended spectrum ß-lactamase found in 80% of cefoxitin-resistant, imipenem-sensitive Bacteroides species tested19. Cefoxitin resistance was observed shortly after its introduction in the 1970s and elements such as Tn4555 may be responsible for the rapid emergence of resistance. More recently, a ß-lactamase gene 98% identical to cfxA was found in 97% of amoxicillin-resistant Prevotella isolated from periodontitis patients20. Since there is potential for Tn4555 and like elements to undermine the effectiveness of antibiotic treatment, it is important to understand all aspects of their dissemination. The mobilizable transposon (MTn), Tn4555 can be transferred (mobilized) to other bacteria when in the presence of CTn, a self-transmissible genetic element that moves from donor genome to recipient genome during cell to cell contact. Although transposons have been defined as mobile genetic elements that randomly insert into DNA by mechanisms not requiring homologous recombination, all know elements including MTns and CTns show some degree of target site selectivity8. CTns appear to have unique target site selection mechanisms which in some cases require homology between 188 the CTn ends (attTn) and the tartget. For example, CTnDOT targets a 10-bp sequence in the Bacteroides choromosome that has homology to its right end15. Tn4555, a clinically relevant MTn, carries all the genes necessary for its insertion into phylogenetically diverse hots. In the investigation of Bacic and Smith4 all bacteria tested, there was preferred insertion site or target and while there were some shared motifs, none of these had ends. A lack of homology suggested that sequence alone was not the main determining factor for site selection, though the translated Bacteroides targets did have homology to BspA from B. forsythus. BspA is a cell-surface-associated, leucine-rich protein involved in adhesion to fibronectin and fibrinogen. BspA has repetitive amino acid sequences suggesting that repetitive areas in the DNA sequence are important for large, imperfect, direct repeats; however, there were no significant repeats in the other Bacteroides targets nor in the E. coli target, and the E. coli target was highly preferred with 90% of all insertions occurring there, more than for any of the others. Also the site of Tn4555 insertion in the original clinical isolate B. vulgatus had no repeats nor did it have any amino acid homology to BspA8. The Mtn share a functional similarity to mobilizable plasmids in that they are not self-transmissible but rather they encode an element-specific mobilization gene(s) and a transfer origin, oriT, that utilize Tcr-element conjugative apparatus to effect their transfer to a new host. These mobilization genes appear to be primarily responsible for DNA processing reactions in which they catalyze a strand-specific nick in the transposon at oriT15. Tn4555 is a mobilizable transposon that carries the cfxA gene, which encodes a clinically significant, broad spectrum ßlactamase responsible for widespread resistance to cefoxitin and other ß-lactams. Initial studies on Tn4555 integration indicated that it proceeds by a site-specific recombination mechanism that utilizes a 6-bp crossover region in the target site20. Clearly Tn4555 is not limited to one or two Bacteroides species or genera in the intestinal niche. There was no difference in excision from preferred sites versus non-preferred sites therefore these targets must benefit insertion5,8. Tn4555 carries antibiotic resistance genes and these elements can be mobilized by a variety of genetic elements can be mobilized by a variety of genetic elements including large Rplasmids or by conjugal transposons such as the large Bacteroides Tc r -elements 5 . However, unlike mobilizable plasmids, there transposons have minimal requerimets for stable maintenance in new hosts. OTHERS CONJUGATIVE TRANSPOSONS Gupta et al.21 used pulsed-field gel electrophoresis to show that the B. uniformis element carrying ermB was integrated into the chromosome and was thus probably a CTn (table 1). The CTn was named CTnBST. CTnBST was estimated to be about 100 kbp in size. A 13-kbp region containing ermB was Rev Esp Quimioter 2011;24 (4): 184-190 28 C. Quesada-Gómez Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates sequenced and compared to sequences then in the databases. The DNA sequence of the ermB gene was identical to genes found in Clostridium difficile. Outside the ermB gene, however, only a few hundred base pairs downstream of ermB had high sequence identity to DNA from gram-positive bacteria22. A Bacteroides CTn, CTnBST, integrates more site specifically than other well-studied CTns, CTnDOT and the enterococcal CTn Tn916. Moreover, the integrase of CTnBST, IntBST, had the C-terminal 6-amino-acid signature that is associated with the catalytic regions of members of the tyrosine recombinase family, most of which integrate site specifically (table 1). Also, in most of these integrases, all of the conserved amino acids are required for integration23. However, the IntBST had that changing three of the six conserved amino acids in the signature, one of which was the presumed catalytic tyrosine, resulted in a 1,000-fold decrease in integration frequency. Also, CTnBST differed from CTnDOT in that whereas the transfer frequency of CTnDOT is stimulated 100- to 1,000fold by tetracycline, CTnBST exhibited constitutive transfer24. CTn341, originally found in a clinical isolate of Bacteroides vulgatus, is 52 kb in size and encodes genes for tetracycline resistance and regulation of transfer, conjugation, and DNA metabolism. The mob region of CTn341 is comprised of three genes, mobABC , as well as the transfer origin, oriT. The oriT region of CTn341 has been located within 100 bp of mobA and the putative nic site was identified within this region. The Mob proteins are predicted to bind at this site and form the relaxosome complex which specifically nicks the DNA at the nick site prior to coupling it to the mating pore apparatus25. The mob gene expression is tightly regulated and transcription is induced by low levels of tetracycline through the two component regulatory system genes rteA-rteB. CTn341, is a member of the most common CTn group, the CTnDOT family26. Sequence analysis revealed 46 genes and one functional group II intron (table 1). The genes fell roughly into three major functional groups: DNA metabolism, regulation and antibiotic resistance, and conjugation 27 . Mutational analyses of genes from each group were used to verify the proposed functional assignments. Based on G+C content and codon usage, the functional groups appeared to belong to different genetic lineages, indicating that CTn341 is a composite, modular element. Comparisons with Bacteroides genome sequences suggested that the basic conjugation and excision genes are conserved in Bacteroides spp27. Other transfer factor is the cLV25. The cLV25 mobilization proteins have similarity to the Tn4399 mobilization proteins and to mobilization proteins encoded by the CTnDOT ermF region. In addition, the genetic organization of the cLV25 mobilization genes and oriT is analogous to those of Tn4399 and the CTnDOT ermF region28 (table 1). Further, cLV25 can functionally crossreact with Tn4399. The cLV25 is a mobilizable transposon based on the presence of imperfect inverted repeats at its termini, an apparent target site repeat, and an open reading frame whose predicted protein has similarity to integrases28. 29 CONCLUSIONS In general, Bacteroides are resistant to a wide variety of (recently many species have acquired resistance to erythromycin, clindamycin, and tetracycline). This high level of antibiotic resistance has prompted concerns that Bacteroides species may become a reservoir for resistance in other, more highly-pathogenic bacterial strains. The conjugation is one of the most important mechanisms in the horizontal transfer of genes in prokaryotes, providing the capacity of variation between species by means of the obtaining of new genetic information. To beginnings of the years 80s the scientific community demonstrated that certain clinical strains of B. fragilis resistant to the antibiotics were capable of transmitting this resistance by means of plasmids and without the presence of these. These elements establish two categories with regard to the resistance of antibiotics: the transfer of the resistance to tetracycline and the transfer of resistance to tetracycline and clindamycin together. The CTn in Bacteroides that can manage to join to the chromosome or plasmids of the receptor bacteria. The potential of the CTn in Bacteroides due to the fact that they are capable of mobilizing plasmids co-residents in trans and in cis, and also of stimulating the excision and the transfer of integrated elements called units not replicate of Bacteroides. 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